Isolated monkey cathepsin S proteins, nucleic acid molecules encoding monkey cathepsin S proteins, and uses thereof

ABSTRACT

The present invention provides isolated monkey Cathepsin S proteins and isolated nucleic acid molecules such as cDNA molecules that encode these monkey Cathepsin S proteins. The present invention specifically provides, for example, isolated monkey Cathepsin S proteins and encoding nucleic acid molecules, methods of producing the monkey Cathepsin S proteins, methods of identifying and screening modulators of the monkey Cathepsin S proteins, and methods of using these modulators to treat a human disease or disorders associated with an orthologous human Cathepsin S protein.

FIELD OF THE INVENTION

The present invention is in the field of cathepsin S proteins,recombinant DNA molecules, and protein production. The present inventionspecifically provides isolated cathepsin S proteins of Cynomologousmonkeys, and isolated nucleic acid molecules encoding such monkeycathepsin S proteins, all of which are useful in the development ofhuman therapeutics and diagnostic compositions and methods.

BACKGROUND OF THE INVENTION

Proteases

In general, proteases typically affect protein cleavage, processing,and/or turnover. Proteases may be categorized into families by thedifferent amino acid sequences (generally between 2 and 10 residues)located on either side of the cleavage site of the protease.

The proper functioning of the cell requires careful control of thelevels of important structural proteins, enzymes, and regulatoryproteins. One of the ways that cells can reduce the steady state levelof a particular protein is by proteolytic degradation. Further, one ofthe ways cells produce functioning proteins is to produce pre- orpro-protein precursors that are processed by proteolytic degradation toproduce an active moiety. Thus, complex and highly regulated mechanismshave been evolved to accomplish this degradation.

Proteases regulate many different cell proliferation, differentiation,and signaling processes by regulating protein turnover and processing.Uncontrolled protease activity (either increased or decreased) has beenimplicated in a variety of disease conditions including inflammation,cancer, arteriosclerosis, and degenerative disorders.

An additional role of intracellular proteolysis is in thestress-response. Cells that are subject to stress such as starvation,heat-shock, chemical insult or mutation respond by increasing the ratesof proteolysis. One function of this enhanced proteolysis is to salvageamino acids from non-essential proteins. These amino acids can then bere-utilized in the synthesis of essential proteins or metabolizeddirectly to provide energy. Another function is in the repair of damagecaused by the stress. For example, oxidative stress has been shown todamage a variety of proteins and cause them to be rapidly degraded.

The International Union of Biochemistry and Molecular Biology (IUBMB)has recommended to use the term peptidase for the subset of peptide bondhydrolases (Subclass E.C 3.4.). The widely used term protease issynonymous with peptidase. Peptidases comprise two groups of enzymes:the endopeptidases and the exopeptidases, which cleave peptide bonds atpoints within the protein and remove amino acids sequentially fromeither N or C-terminus respectively. The term proteinase is also used asa synonym word for endopeptidase and four mechanistic classes ofproteinases are recognized by the IUBMB: two of these are describedbelow (also see: Handbook of Proteolytic Enzymes by Barrett, Rawlings,and Woessner AP Press, NY 1998). Also, for a review of the various usesof proteases as drug targets, see: Weber M, Emerging treatments forhypertension: potential role for vasopeptidase inhibition; Am JHypertens 1999 November;12(11 Pt 2):139S–147S; Kentsch M, Otter W, Novelneurohormonal modulators in cardiovascular disorders. The therapeuticpotential of endopeptidase inhibitors, Drugs R D 1999 April;1(4):331–8;Scarborough R M, Coagulation factor Xa: the prothrombinase complex as anemerging therapeutic target for small molecule inhibitors, J Enzym Inhib1998;14(1):15–25; Skotnicki J S, et al., Design and syntheticconsiderations of matrix metalloproteinase inhibitors, Ann N Y Acad Sci1999 Jun. 30;878:61–72; McKerrow J H, Engel J C, Caffrey C R, Cysteineprotease inhibitors as chemotherapy for parasitic infections, Bioorg MedChem 1999 April;7(4):639–44; Rice K D, Tanaka R D, Katz B A, Numerof RP, Moore W R, Inhibitors of tryptase for the treatment of mastcell-mediated diseases, Curr Pharm Des 1998 October;4(5):381–96;Materson B J, Will angiotensin converting enzyme genotype, receptormutation identification, and other miracles of molecular biology permitreduction of NNT Am J Hypertens 1998 August;11(8 Pt 2):138S–142S.

Cathepsin S

Cathepsin S is a lysosomal cysteine protease that catalyzes the removalof the invariant chain (Ii) from MHC class II molecules (Morton, P. A.,Zacheis, M. L., Giacoletto, K. S., Manning, J. A., Schwartz, B. D.,(1995) J. Immunol. 154, 137–150). Ii serves dual functions. Ii serves asa molecular chaperon that promotes the MHC class II/Ii complex exit fromthe ER to the endosomal system through the secretory pathway. Ii alsofunctions as a molecular inhibitor that occupies the peptide-bindingpocket of MHC class II to prevent premature binding of native peptidesin the secretory pathway (Hsieh, C. S., deRoos, P., Honey, K., Beers,C., and Rudensky, A. Y. (2002) J. Immunol. 168, 2618–2625). Experimentsusing protease inhibitors and knockout mice have clearly shown thatcathepsin S is involved in proteolytic clearance of Ii from MHC class IImolecules (Riese, R. J., Mitchell, R. N., Villadangos, J. A., Shi, G.P., Palmer, J. T., Karp, E. R., De Sanctis, G. T., Ploegh, H. L., andChapman, H. A. (1998) J. Clin. Invest. 101, 2351–2363; Nakagawa, T. Y.,Brissette, E. H., Lira, P. D., Griffiths, R. J., Petrushova, N., Stock,J., McNeish, J. D., Eastman, S. E., Howard, E. D., Clarke, S. R.,Rosloniec, E. F., Elliott, E. A., and Rudensky, A. Y. (1999) Immunity10, 207–217). Inhibition of cathepsin S precludes antigen loading to MHCclass II molecules and disables the antigen-presenting cell, therebypreventing the antigen-presenting cell from presenting antigen to CD4+ Tcells (Shi, G.-P., Villadangos, J. A., Dranoff, G., Small, C., Gu, L.,Haley, K. J., Rises, R., Ploegh, H. L., Chapman, H. A. (1999) Immunity10, 197–206). Selective inhibition of cathepsin S is thereforeconsidered to be therapeutically useful to attenuate the elevated immuneresponses found in many autoimmune disorders.

To investigate the effectiveness of cathepsin S inhibitors, it istypically necessary to evaluate species-specific cathepsin S activity indifferent animal autoimmune disease models. In particular, to developcathepsin S inhibitors for the treatment of human disorders, it istypically desirable to test candidate inhibitor compounds againstcathepsin S proteins of non-human primates, such as monkeys. Non-humanprimates, because they are more closely related to humans than rodentsand other laboratory animals, can more accurately predict theeffectiveness (as well as toxicity and other undesirable side effects)of the inhibitor compound in humans, and are therefore advantageous foruse as animal models for evaluating the use of potential therapeuticcompounds for treating human disease, prior to administration of thecompounds to humans.

Consequently, because cathepsin S proteins are well established in theart as playing key roles in important human diseases (such as autoimmunedisorders), and because monkeys serve as an optimal animal model forevaluating candidate therapeutic compounds for the treatment of humandiseases, a need exists in the art for isolated cathepsin S proteins,and encoding nucleic acid molecules, from non-human primates such asmonkeys.

Proteases and Cancer

Proteases are critical elements at several stages in the progression ofmetastatic cancer. In this process, the proteolytic degradation ofstructural protein in the basal membrane allows for expansion of a tumorin the primary site, evasion from this site as well as homing andinvasion in distant, secondary sites. Also, tumor induced angiogenesisis required for tumor growth and is dependent on proteolytic tissueremodeling. Transfection experiments with various types of proteaseshave shown that the matrix metalloproteases play a dominant role inthese processes in particular gelatinases A and B (MMP-2 and MMP-9,respectively). For an overview of this field, see Mullins, et al.,Biochim. Biophys. Acta 695, 177, 1983; Ray, et al., Eur. Respir. J. 7,2062, 1994; Birkedal-Hansen, et al., Crit. Rev. Oral Biol. Med. 4, 197,1993.

Furthermore, it was demonstrated that inhibition of degradation ofextracellular matrix by the native matrix metalloprotease inhibitorTIMP-2 (a protein) arrests cancer growth (DeClerck, et al., Cancer Res.52, 701, 1992) and that TIMP-2 inhibits tumor-induced angiogenesis inexperimental systems (Moses, et al. Science 248, 1408, 1990). For areview, see DeClerck, et al., Ann. N. Y. Acad. Sci. 732, 222, 1994. Itwas further demonstrated that the synthetic matrix metalloproteaseinhibitor batimastat when given intraperitoneally inhibits human colontumor growth and spread in an orthotopic model in nude mice (Wang, etal. Cancer Res. 54, 4726, 1994) and prolongs the survival of micebearing human ovarian carcinoma xenografts (Davies, et. al., Cancer Res.53, 2087, 1993). The use of this and related compounds has beendescribed in Brown, et al., WO-9321942 A2.

There are several patents and patent applications claiming the use ofmetalloproteinase inhibitors for the retardation of metastatic cancer,promoting tumor regression, inhibiting cancer cell proliferation,slowing or preventing cartilage loss associated with osteoarthritis orfor treatment of other diseases as noted above (e.g. Levy, et al.,WO-9519965 A1; Beckett, et al., WO-9519956 A1; Beckett, et al.,WO-9519957 A1; Beckett, et al., WO-9519961 A1; Brown, et al., WO-9321942A2; Crimmin, et al., WO-9421625 A1; Dickens, et al., U.S. Pat. No.4,599,361; Hughes, et al., U.S. Pat. No. 5,190,937; Broadhurst, et al.,EP 574758 A1; Broadhurst, et al., EP 276436; and Myers, et al., EP520573 A1.

Mammalian Models of Human Disease

Non-human primates such as monkeys, as well as rodents such as mice (Musmusculus) and other animals such as rabbits and guinea pigs, arecommonly used in biomedical research as model systems for studying humandiseases and for developing therapeutic and diagnostic agents for humandiseases. Detailed descriptions of techniques and protocols formanipulating and using such animal models of human disease, particularlymouse models, such as techniques for using homologous recombination toproduce mutant mouse strains and mutant cell lines with specific genesinactivated (e.g., “knockout” mice), are readily available, and thetechniques described for mice can be applied to monkeys and othernon-human primates as well as other laboratory animals. For example, fora review of mouse models for studying gene/protein functions andinteractions, cell biology, and human diseases, including mousemutagenesis techniques currently used in the art, see Current Protocolsin Molecular Biology, John Wiley & Sons, Inc., “Manipulating the MouseGenome”, chapter 23, supplements 51–53 (2000–2001); Nolan et al., NatGenet 2000 August;25(4):440–3; Brown, J Inherit Metab Dis 1998August;21(5):532–9; Nolan, Pharmacogenomics 2000 August; 1(3):243–55;Justice, Nat Rev Genet 2000 November; 1(2): 109–15; and Mansuy et al.,Exp Physiol 2000 November;85(6):661–79.

Protease proteins, particularly cathepsin S proteases, are a majortarget for drug action and development, and the monkey provides anoptimal animal model in which to study these protease proteins and thediseases with which they are associated. Accordingly, it is valuable tothe field of pharmaceutical development to identify and characterizepreviously unknown members of this subfamily of protease proteins in themonkey. The present invention advances the state of the art by providingisolated monkey cathepsin S proteins and encoding nucleic acidmolecules.

SUMMARY OF THE INVENTION

The present invention is based in part on the cloning, expression, andisolation of monkey cathepsin S polypeptides and encoding nucleic acidmolecules such as cDNA molecules. These unique monkey polypeptides, andnucleic acid molecules that encode these polypeptides, are useful, forexample, in the development, screening, and evaluation of humantherapeutic agents (e.g., small molecule compounds, antibodies,therapeutic proteins, nucleic acid agents such as RNAi or antisenseagents, etc.), particularly therapeutic agents that target cathepsin S,such as cathepsin S inhibitors.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides the nucleotide sequence of a cDNA molecule (SEQ ID NO:1)that encodes a Cynomologous monkey cathepsin S polypeptide (SEQ IDNO:2).

FIGS. 2A–2B (collectively referred to herein as FIG. 2) provide analignment of cathepsin S amino acid sequences from Cynomologous monkey(SEQ ID NO:2), monkey sml cells (Saimiri boliviensis) (SEQ ID NO:3),human (Genbank accession number BC002642.2) (SEQ ID NO:4), dog (canisfamiliaris; Genbank accession number AY156692) (SEQ ID NO:5), and mouse(Mus musculus; Genbank gi3850787) (SEQ ID NO:6). A consensus sequence(SEQ ID NO:7) is also shown below these five sequences.

FIG. 3 provides an image of a Gelcode stained gel showing purificationof monkey cathepsin S.

DETAILED DESCRIPTION OF THE INVENTION

General Description

The present invention is based on the cloning, expression, andpurification/isolation of cathepsin S proteins (cathepsin S may beinterchangeable referred to as CatS) from Cynomologous monkeys.Cathepsin S is a lysosomal cysteine protease. The present inventionprovides amino acid sequences of Cynomologous monkey cathepsin Sproteins, and nucleic acid molecules such as cDNA sequences that encodethese monkey cathepsin S proteins. The present invention also providesmethods of expressing/producing and isolating the monkey cathepsin Sproteins, methods of identifying modulators of the monkey cathepsin Sproteins (such as by using the monkey cathepsin S protein in screeningassays to screen candidate compounds that target the cathepsin Sprotein, such as cathepsin S inhibitors), and methods of using thesemodulators to treat human disease or disorders associated with anorthologous human cathepsin S protein.

The 1.7 kb full-length Cynomologous cathepsin S cDNA, which is shown inFIG. 1 as SEQ ID NO:1, shares 96.3% overall nucleotide sequence identitywith the human cathepsin S cDNA. The complete Cynomologous monkeypre-pro-CatS protein is a 331 amino acid protein with 96.7% identity tothat of human (an alignment comparing cathepsin S amino acid sequencesfrom multiple different species is shown in FIG. 2). There are ten aminoacid sequence substitutions compared to the human protein sequence (seeFIG. 2). Three of the ten amino acid substitutions are in the “pre-pro”region of the protein that is cleaved off and does not become part ofthe “mature” CatS. The other seven amino acid substitutions occur in themature CatS sequence, and two of these seven substitutions are predictedto affect the enzyme structure and/or interactions between the enzymeand compounds such as CatS inhibitor compounds. Specifically, an Arg toSer substitution exists at residue 256, which forms the S1′ pocket, anda Thr to Arg substitution exists at residue 188, which is expected toaffect the structure of the S2 and S3 pockets. The other eight aminoacid substitutions are (listed in the format of: monkey aminoacid/residue number/human amino acid): Q3R, A111P, Q113W, N197K, T210M,V242A, V288D, and R303H.

The monkey pro-CatS can be expressed and purified from yeast (see FIG.3) and the enzyme is fully active after the pro-peptide is processedauto-catalytically in vitro. The availability of significant quantitiesof active CatS will facilitate the understanding of CatS interactionswith different inhibitor compounds and will facilitate the evaluation ofinhibitor compounds in a monkey disease model.

In FIG. 1, the diamond symbol at nucleotide151 represents the cleavagesite for the pre-sequence. Thus, the amino acid sequence of thepro-sequence begins with leucine (“L”), histidine (“H”), lysine (“K”).The down-arrow symbol at nucleotide 443 represents the cleavage site forthe pro-sequence. Thus, the amino acid sequence of the mature enzymebegins with leucine (“L”), proline (“P”), aspartic acid (“D”).

The peptides that are provided by the present invention are useful forthe development of commercially important products and services, forexample. The art has clearly established the commercial importance ofcathepsin S proteins. Some of the more specific features of the peptidesof the present invention, and the uses thereof, are described herein,particularly in the Background of the Invention and/or are known withinthe art for cathepsin S proteins.

As used herein, the terms “subject(s)”, “individual(s)”, and“patient(s)” may refer to monkeys or other non-human primates, humans,or any other animal. The methods and compositions provided by thepresent invention, particularly the monkey protease proteins andencoding nucleic acid molecules, are especially useful for studyinghuman diseases and for developing drugs/therapeutic agents in a monkey(or other non-human primate) model in order to treat humandiseases/disorders and to evaluate candidate therapeutic compounds.However, the present invention is not so limited and may also be usefulfor studying diseases/disorders in a monkey model that affect any animaland for developing therapeutic treatments for use in any animal. Forexample, the agents of the present invention may be used in a monkeymodel to develop veterinary pharmaceutical/therapeutic agents fortreating diseases that affect domesticated animals and/or commerciallyvaluable livestock such as cows and pigs. Additionally, it would beapparent to one of ordinary skill in the art that the compositions ofthe present invention may also be used in other animal model systems,particularly in those closely related to the Cynomologous monkey, suchas other monkey species and other non-human primates.

Specific Embodiments

Peptide Molecules

The present invention provides nucleic acid molecules that encode monkeycathepsin S proteins (protein and cDNA sequences are provided in FIG.1). The protein sequences provided in FIG. 1, as well as the obviousvariants described herein, particularly allelic variants as identifiedherein and using the information in FIG. 1, will be referred to hereinas the cathepsin S peptides/proteins, protease peptides/proteins, orpeptides/proteins of the present invention. The terms “protein”,“peptide”, and “polypeptide” are used herein interchangeably.

The present invention provides isolated peptide and protein moleculesthat consist of, consist essentially of, or comprise the amino acidsequence of the cathepsin S protein disclosed in FIG. 1 (encoded by thecDNA sequence which is also shown in FIG. 1), as well as all obviousvariants of these proteins that are within the art to make and use. Someof these variants are described in detail below.

As used herein, a peptide is said to be “isolated” or “purified” when itis substantially free of cellular material or free of chemicalprecursors or other chemicals. The peptides of the present invention canbe purified to homogeneity or other degrees of purity. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the peptide,even if in the presence of considerable amounts of other components (thefeatures of an isolated nucleic acid molecule is discussed below).

In some uses, “substantially free of cellular material” includespreparations of the peptide having less than about 30% (by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the peptide is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of the peptide in which it is separatedfrom chemical precursors or other chemicals that are involved in itssynthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of theprotease peptide having less than about 30% (by dry weight) chemicalprecursors or other chemicals, less than about 20% chemical precursorsor other chemicals, less than about 10% chemical precursors or otherchemicals, or less than about 5% chemical precursors or other chemicals.

The isolated protease peptide can be purified from cells that naturallyexpress it, purified from cells that have been altered to express it(recombinant), or synthesized using known protein synthesis methods. Forexample, a nucleic acid molecule encoding the protease peptide is clonedinto an expression vector, the expression vector introduced into a hostcell and the protein expressed in the host cell. The protein can then beisolated from the cells by an appropriate purification scheme usingstandard protein purification techniques. Many of these techniques aredescribed in detail below.

Accordingly, the present invention provides proteins that consist of theamino acid sequence provided in FIG. 1 (SEQ ID NO:2), for example,proteins encoded by the cDNA nucleic acid sequence shown in FIG. 1 (SEQID NO:1). The amino acid sequence of such a protein is provided inFIG. 1. A protein consists of an amino acid sequence when the amino acidsequence is the final amino acid sequence of the protein.

The present invention further provides proteins that consist essentiallyof the amino acid sequence provided in FIG. 1 (SEQ ID NO:2), forexample, proteins encoded by the cDNA nucleic acid sequence shown inFIG. 1 (SEQ ID NO:1). A protein consists essentially of an amino acidsequence when such an amino acid sequence is present with only a fewadditional amino acid residues, for example from about 1 to about 100 orso additional residues, typically from 1 to about 20 additional residuesin the final protein.

The present invention further provides proteins that comprise the aminoacid sequence provided in FIG. 1 (SEQ ID NO:2), for example, proteinsencoded by the cDNA nucleic acid sequence shown in FIG. 1 (SEQ ID NO:1).A protein comprises an amino acid sequence when the amino acid sequenceis at least part of the final amino acid sequence of the protein. Insuch a fashion, the protein can be only the peptide or have additionalamino acid molecules, such as amino acid residues (contiguous encodedsequence) that are naturally associated with it or heterologous aminoacid residues/peptide sequences. Such a protein can have a fewadditional amino acid residues or can comprise several hundred or moreadditional amino acids. The preferred classes of proteins that arecomprised of the protease peptides of the present invention are thenaturally occurring mature proteins. A brief description of how varioustypes of these proteins can be madelisolated is provided below.

The protease peptides of the present invention can be attached toheterologous sequences to form chimeric or fusion proteins. Suchchimeric and fusion proteins comprise a protease peptide operativelylinked to a heterologous protein having an amino acid sequence notsubstantially homologous to the protease peptide. “Operatively linked”indicates that the protease peptide and the heterologous protein arefused in-frame. The heterologous protein can be fused to the N-terminusor C-terminus of the protease peptide.

In some uses, the fusion protein does not affect the activity of theprotease peptide per se. For example, the fusion protein can include,but-is:not limited to, enzymatic fusion proteins, for examplebeta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-Hisfusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins,particularly poly-His fusions, can facilitate the purification ofrecombinant protease peptide. In certain host cells (e.g., mammalianhost cells), expression and/or secretion of a protein can be increasedby using a heterologous signal sequence.

A chimeric or fusion protein can be produced by standard recombinant DNAtechniques. For example, DNA fragments coding for the different proteinsequences are ligated together in-frame in accordance with conventionaltechniques. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PC R amplification of gene fragments can be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive gene fragments which can subsequently be annealed andre-amplified to generate a chimeric gene sequence (see Ausubel et al.,Current Protocols in Molecular Biology, 1992). Moreover, many expressionvectors are commercially available that already encode a fusion moiety(e.g., a OST protein). A protease peptide-encoding nucleic acid can becloned into such an expression vector such that the fusion moiety islinked in-frame to the protease peptide.

As mentioned above, the present invention also provides and enablesobvious variants of the amino acid sequence of the proteins of thepresent invention, such as naturally occurring mature forms of thepeptide, allelic/sequence variants of the peptides, non-naturallyoccurring recombinantly derived variants of the peptides, and orthologsand paralogs of the peptides. Such variants can readily be generatedusing art-known techniques in the fields of recombinant nucleic acidtechnology and protein biochemistry. It is understood, however, thatvariants exclude any amino acid sequences disclosed prior to theinvention.

Such variants can readily be identified/made using molecular techniquesand the sequence information disclosed herein. Further, such variantscan readily be distinguished from other peptides based on sequenceand/or structural homology to the protease peptides of the presentinvention. The degree of homology/identity present will be basedprimarily on whether the peptide is a functional variant ornon-functional variant, the amount of divergence;present in the paralogfamily and the evolutionary distance between the orthologs.

To determine the percent identity of two amino acid sequences or twonucleic acid sequences, the sequences are aligned for optimal-comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% ormore of the length of a reference sequence is aligned for comparisonpurposes. The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, then the molecules are identical at that position (as usedherein amino acid or nucleic acid “identity” is equivalent to amino acidor nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

The comparison of sequences and determination of percent identity andsimilarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). In a preferred embodiment, the percent identity betweentwo amino acid sequences is determined using the Needleman and Wunsch(J. Mol. Biol. (48):444–453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package, usingeither a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16,14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. Inyet another preferred embodiment, the percent identity between twonucleotide sequences is determined using the GAP program in the GCGsoftware package (Devereux, J., et al., Nucleic Acids Res. 12(1):387(1984)), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60,70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In anotherembodiment, the percent identity between two amino acid or nucleotidesequences is determined using the algorithm of E. Myers and W. Miller(CABIOS, 4:11–17 (1989)) which has been incorporated into the ALIGNprogram (version 2.0), using a PAM120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol.215:403–10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17):3389–3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

Full-length pre-processed forms, as well as mature processed forms, ofproteins that comprise one of the peptides of the present invention canreadily be identified as having complete sequence identity to one of theprotease peptides of the present invention as well as being encoded bythe same genetic locus as the protease peptide provided herein.

Allelic variants of a protease peptide can readily be identified asbeing a monkey protein having a high degree (significant) of sequencehomology/identity to at least a portion of the protease peptide as wellas being encoded by the same genetic locus as the protease peptideprovided herein. As used herein, two proteins (or a region of theproteins) have significant homology when the amino acid sequences aretypically at least about 70–80%, 80–90%, and more typically at leastabout 90–95, 96, 97, 98, or 99% homologous. A significantly homologousamino acid sequence, according to the present invention, will be encodedby a nucleic acid sequence that will hybridize to a protease peptideencoding nucleic acid molecule under stringent conditions as more fullydescribed below.

Paralogs of a protease peptide can readily be identified as having somedegree of significant sequence homology/identity to at least a portionof the protease peptide, as being encoded by a monkey gene, and ashaving similar activity or function. Two proteins will typically beconsidered paralogs when the amino acid sequences are typically at leastabout 60% or greater, and more typically at least about 70% or greaterhomology through a given region or domain. Such paralogs will be encodedby a nucleic acid sequence that will hybridize to a protease peptideencoding nucleic acid molecule under moderate to stringent conditions asmore fully described below.

Orthologs of a protease peptide can readily be identified as having somedegree of significant sequence homology/identity to at least a portionof the monkey protease peptide as well as being encoded by a gene fromanother organism. Preferred orthologs will be isolated from mammals,preferably humans or other primates, for the development of humantherapeutic targets and agents. Such orthologs will be encoded by anucleic acid sequence that will hybridize to a monkey protease-encodingnucleic acid molecule under moderate to stringent conditions, as morefully described below, depending on the degree of relatedness of the twoorganisms yielding the proteins.

Non-naturally occurring variants of the protease peptides of the presentinvention can readily be generated using recombinant techniques. Suchvariants include, but are not limited to deletions, additions andsubstitutions in the amino acid sequence of the protease peptide. Forexample, one class of substitutions are conserved amino acidsubstitution. Such substitutions are those that substitute a given aminoacid in a protease peptide by another amino acid of likecharacteristics. Typically seen as conservative substitutions are thereplacements, one for another, among the aliphatic amino acids Ala, Val,Leu, and Ile; interchange of the hydroxyl residues Ser and Thr; exchangeof the acidic residues Asp and Glu; substitution between the amideresidues Asn and Gln; exchange of the basic residues Lys and Arg; andreplacements among the aromatic residues Phe and Tyr. Guidanceconcerning which amino acid changes are likely to be phenotypicallysilent are found in Bowie et al., Science 247:1306–1310 (1990).

Variant protease peptides can be fully functional or can lack functionin one or more activities, e.g. ability to bind substrate, ability tocleave substrates ability to participate in a signaling pathway, etc.Fully functional variants typically contain only conservative variationor variation in non-critical residues or in noncritical regions.Functional variants can also contain substitution of similar amino acidsthat result in no change or an insignificant change in function.Alternatively, such substitutions may positively or negatively affectfunction to some degree.

Non-functional variants typically contain one or more non-conservativeamino acid substitutions, deletions, insertions, inversions, ortruncation or a substitution, insertion, inversion, or deletion in acritical residue or critical region.

Amino acids that are essential for function can be identified by methodsknown in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham et al., Science 244:1081–1085 (1989)), and canalso be predicted based on the alignment of cathepsin S amino acidsequences from multiple species, as shown in FIG. 2 (since amino acidresidues that are more conserved across species may typically bepredicted to be more likely to have a functional affect).Alanine-scanning mutagenesis introduces single alanine mutations atevery residue in the molecule. The resulting mutant molecules are thentested for biological activity such as protease activity or in assayssuch as an in vitro proliferative activity. Sites that are critical forbinding partner/substrate binding can also be determined by structuralanalysis such as crystallization, nuclear magnetic resonance orphotoaffinity labeling (Smith et al., J. Mol. Biol. 224:899–904 (1992);de Vos et al. Science 255:306–312 (1992)).

The present invention further provides fragments of the proteasepeptides, in addition to proteins and peptides that comprise and consistof such fragments. The fragments to which the invention pertains,however, are not to be construed as encompassing fragments that may bedisclosed publicly prior to the present invention.

As used herein, a fragment comprises at least 8, 10, 12, 14, 16, or morecontiguous amino acid residues from a protease peptide. Such fragmentscan be chosen based on the ability to retain one or more of thebiological activities of the protease peptide or could be chosen for theability to perform a function, e.g. bind a substrate or act as animmunogen. Particularly important fragments are biologically activefragments, peptides that are, for example, about 8 or more amino acidsin length. Such fragments will typically comprise a domain or motif ofthe protease peptide, e.g., active site, a transmembrane domain or asubstrate-binding domain. Further, possible fragments include, but arenot limited to, domain or motif containing fragments, soluble peptidefragments, and fragments containing immunogenic structures. Predicteddomains and functional sites are readily identifiable by computerprograms well known and readily available to those of skill in the art(e.g., PROSITE analysis).

Polypeptides often contain amino acids other than the 20 amino acidscommonly referred to as the 20 naturally occurring amino acids. Further,many amino acids, including the terminal amino acids, may be modified bynatural processes, such as processing and other post-translationalmodifications, or by chemical modification techniques well known in theart. Common modifications that occur naturally in protease peptides aredescribed in basic texts, detailed monographs, and the researchliterature, and they are well known to those of skill in the art.

Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

Such modifications are well known to those of skill in the art and havebeen described in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in most basic texts,such as Proteins—Structure and Molecular Properties, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993). Many detailedreviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1–12 (1983); Seifter et al. (Meth. Enzymol.182: 626–646 (1990)) and Rattan et al. (Ann. N.Y. Acad. Sci. 663:48–62(1992)).

Accordingly, the protease peptides of the present invention alsoencompass derivatives or analogs in which a substituted amino acidresidue is not one encoded by the genetic code, in which a substituentgroup is included, in which the mature protease peptide is fused withanother compound, such as a compound to increase the half-life of theprotease peptide (for example, polyethylene glycol), or in which theadditional amino acids are fused to the mature protease peptide, such asa leader or secretory sequence or a sequence for purification of themature protease peptide or a pro-protein sequence.

Protein/Peptide Uses

The proteins of the present invention can be used in substantial andspecific assays; to raise antibodies or to elicit another immuneresponse; as a reagent (including the labeled reagent) in assaysdesigned to quantitatively determine levels of the protein (or itsbinding partner or ligand) in biological fluids; and as markers fortissues in which the corresponding protein is preferentially expressed(either constitutively or at a particular stage of tissuedifferentiation or development or in a disease state). Where the proteinbinds or potentially binds to another protein or ligand (such as, forexample, in a protease-effector protein interaction or protease-ligandinteraction), the protein can be used to identify the bindingpartner/ligand so as to develop a system to identify inhibitors of thebinding interaction. Any or all of these uses are capable of beingdeveloped into reagent grade or kit format for commercialization ascommercial products.

Methods for performing the uses listed above are well known to thoseskilled in the art. References disclosing such methods include“Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring HarborLaboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds.,1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

The potential uses of the peptides of the present invention are basedprimarily on the source of the protein as well as the class/action ofthe protein. For example, proteases isolated from the monkey, and theirhuman/mammalian orthologs, serve as targets for identifying agents foruse in mammalian therapeutic applications, e.g. a human drug,particularly in modulating a biological or pathological response in acell or tissue that expresses the protease. A large percentage ofpharmaceutical agents are being developed that modulate the activity ofprotease proteins, particularly cathepsin S proteases (see Background ofthe Invention). The structural and functional information provided inthe Background and Figures provide specific and substantial uses for themolecules of the present invention. Such uses can readily be determinedusing the information provided herein, that which is known in the art,and routine experimentation.

The proteins of the present invention (including variants and fragmentsthat may have been disclosed prior to the present invention) are usefulin biological assays to evaluate cathepsin S proteases and/or compoundsthat target cathepsin S proteases, such as cathepsin S inhibitors. Suchassays typically involve any of the known protease functions oractivities or properties useful for diagnosis and treatment ofprotease-related conditions, especially functions or activities orproperties specific to cathepsin S proteases, particularly in cells andtissues that express the protease.

The proteins of the present invention are also useful in drug screeningassays, in cell-based or cell-free systems. Cell-based systems can benative, i.e., cells that normally express the protease, as a biopsy orexpanded in cell culture. In an alternate embodiment, cell-based assaysinvolve recombinant host cells expressing the protease protein.

The polypeptides can be used to identify compounds that modulateprotease activity of the protein in its natural state or an altered formthat causes a specific disease or pathology associated with theprotease. Both the proteases of the present invention and appropriatevariants and fragments can be used in high-throughput screens to assaycandidate compounds for the ability to bind to the protease. Thesecompounds can be further screened against a functional protease todetermine the effect of the compound on the protease activity. Further,these compounds can be tested in a monkey or other animal orinvertebrate systems to determine activity/effectiveness. Compounds canbe identified that activate (agonist) or inactivate (antagonist) theprotease to a desired degree.

Further, the proteins of the present invention can be used to screen acompound for the ability to stimulate or inhibit interaction between theprotease protein and a molecule that normally interacts with theprotease protein, e.g. a substrate or a component of the signal pathwaythat the protease protein normally interacts (for example, a protease).Such assays typically include the steps of combining the proteaseprotein with a candidate compound under conditions that allow theprotease protein, or fragment, to interact with the target molecule, andto detect the formation of a complex between the protein and the targetor to detect the biochemical consequence of the interaction with theprotease protein and the target, such as any of the associated effectsof signal transduction such as protein cleavage, cAMP turnover, andadenylate cyclase activation, etc.

Candidate compounds include, for example, 1) peptides such as solublepeptides, including Ig-tailed fusion peptides and members of randompeptide libraries (see, e.g., Lam et al., Nature 354:82–84 (1991);Houghten et al., Nature 354:84–86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767–778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

One candidate compound is a soluble fragment of the receptor thatcompetes for substrate binding. Other candidate compounds include mutantproteases or appropriate fragments containing mutations that affectprotease function and thus compete for substrate. Accordingly, afragment that competes for substrate, for example with a higheraffinity, or a fragment that binds substrate but does not allow release,is encompassed by the invention.

The invention further includes other end point assays to identifycompounds that modulate (stimulate or inhibit) protease activity. Theassays typically involve an assay of events in the signal transductionpathway that indicate protease activity. Thus, the cleavage of asubstrate, inactivation/activation of a protein, a change in theexpression of genes that are up- or down-regulated in response to theprotease protein dependent signal cascade can be assayed.

Any of the biological or biochemical functions mediated by the proteasecan be used as an endpoint assay. These include all of the biochemicalor biochemical/biological events described herein, in the referencescited herein, incorporated by reference for these endpoint assaytargets, and other functions known to those of ordinary:skill in the artor that can be readily identified using the information provided herein.Specifically, a biological function of a cell or tissues that expressesthe protease can be assayed.

Binding and/or activating compounds can also be screened by usingchimeric protease proteins in which the amino terminal extracellulardomain, or parts thereof, the entire transmembrane domain or subregions,such as any of the seven transmembrane segments or any of theintracellular or extracellular loops and the carboxy terminalintracellular domain, or parts thereof, can be replaced by heterologousdomains or subregions. For example, a substrate-binding region can beused that interacts with a different substrate than that which isrecognized by the native protease. Accordingly, a different set ofsignal transduction components is available as an end-point assay foractivation. This allows for assays to be performed in other than thespecific host cell from which the protease is derived.

The proteins of the present invention are also useful in competitionbinding assays in methods designed to discover compounds that interactwith the protease (e.g. binding partners and/or ligands). Thus, acompound is exposed to a protease polypeptide under conditions thatallow the compound to bind or to otherwise interact with thepolypeptide. Soluble protease polypeptide is also added to the mixture.If the test compound interacts with the soluble protease polypeptide, itdecreases the amount of complex formed or activity from the proteasetarget. This type of assay is particularly useful in cases in whichcompounds are sought that interact with specific regions of theprotease. Thus, the soluble polypeptide that competes with the targetprotease region is designed to contain peptide sequences correspondingto the region of interest.

To perform cell free drug screening assays, it is sometimes desirable toimmobilize either the protease protein, or fragment, or its targetmolecule to facilitate separation of complexes from uncomplexed forms ofone or both of the proteins, as well as to accommodate automation of theassay.

Techniques for immobilizing proteins on matrices can be used in the drugscreening assays. In one embodiment, a fusion protein can be providedwhich adds a domain that allows the protein to be bound to a matrix. Forexample, glutathione-S-transferase fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then-combined withthe cell lysates (e.g., ³⁵S-labeled) and the candidate compound, and themixture incubated under conditions conducive to complex fofoation (e.g.,at physiological conditions for salt and pH). Following incubation, thebeads are washed to remove any unbound label, and the matrix immobilizedand radiolabel determined directly, or in the supernatant after thecomplexes are dissociated. Alternatively, the complexes can bedissociated from the matrix, separated by SDS-PAGE, and the level ofprotease-binding protein found in the bead fraction quantitated from thegel using standard electrophoretic techniques. For example, either thepolypeptide or its target molecule can be immobilized utilizingconjugation of biotin and streptavidin using techniques well known inthe art. Alternatively, antibodies reactive with the protein but whichdo not interfere with binding of the protein to its target molecule canbe derivatized to the wells of the plate, and the protein trapped in thewells by antibody conjugation. Preparations of a protease-bindingprotein and a candidate compound are incubated in the proteaseprotein-presenting wells and the amount of complex trapped in the wellcan be quantitated. Methods for detecting such complexes, in addition tothose described above for the GST-immobilized complexes, includeimmunodetection of complexes using antibodies reactive with the proteaseprotein target molecule, or which are reactive with protease protein andcompete with the target molecule, as well as enzyme-linked assays whichrely on detecting an enzymatic activity associated with the targetmolecule.

Agents that modulate one of the proteases of the present invention canbe identified using one or more of the above assays, alone or incombination. It is generally preferable to use a cell-based or cell freesystem first and then confirm activity in a monkey or other animal modelsystem. Such model systems are well known in the art and can readily beemployed in this context.

Modulators of protease protein activity identified according to thesedrug-screening assays can be used to treat a subject with a disordermediated by the protease pathway, by treating cells or tissues thatexpress the protease. These methods of treatment include the steps ofadministering a modulator of protease activity in a pharmaceuticalcomposition to a subject in need of such treatment, the modulator beingidentified as described herein. The subject is typically a human, butmay be any other vertebrate or invertebrate animal that one desires totreat using a compound identified-according to the screening assaysprovided herein that utilize the monkey protease proteins/genes of thepresent invention. For example, the subject may be a commerciallyvaluable or domesticated animal, such as a cow, pig, horse, etc.

In yet another aspect of the invention, the protease proteins can beused as “bait proteins” in a two-hybrid assay or three-hybrid assay(see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223–232; Madura et al. (1993) J. Biol. Chem. 268:12046–12054; Bartelet al. (1993) Biotechniques 14:920–924; Iwabuchi et al. (1993) Oncogene8:1693–1696; and Brent WO94/10300), to identify other proteins, whichbind to or interact with the protease and are involved in proteaseactivity. Such protease-binding proteins are also likely to be involvedin the propagation of signals by the protease proteins or proteasetargets as, for example, downstream elements of a protease-mediatedsignaling pathway. Alternatively, such protease-binding proteins arelikely to be protease inhibitors.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a protease proteinis fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming aprotease-dependent complex, the DNA-binding and activation domains ofthe transcription factor are brought into close proximity. Thisproximity allows transcription of a reporter gene (e.g., LacZ) which isoperably linked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with the protease protein.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein ina monkey or other appropriate animal model. For example, an agentidentified as described herein (e.g., a protease-modulating agent, anantisense protease nucleic acid molecule, a protease-specific antibody,or a protease-binding partner) can be used in a monkey or other animalmodel to determine the efficacy, toxicity, or side effects of treatmentwith such an agent. Alternatively, an agent identified as describedherein can be used in a monkey or other animal model to determine themechanism of action of such an agent. Furthermore, this inventionpertains to uses of novel agents identified by the above-describedscreening assays for treatments as described herein.

The protease proteins of the present invention are also useful toprovide a target for diagnosing a disease or predisposition to diseasemediated by the peptide. Accordingly, the invention provides methods fordetecting the presence, or levels of, the protein (or encoding mRNA) ina cell, tissue, or organism. The method typically involves contacting abiological sample with a compound capable of interacting with theprotease protein such that the interaction can be detected. Such anassay can be provided in a single detection format or a multi-detectionformat such as an antibody chip array.

One agent for detecting a protein in a sample is an antibody capable ofselectively binding to protein. A biological sample includes tissues,cells and biological fluids isolated from a subject, as well as tissues,cells and fluids present within a subject.

The peptides of the present invention also provide targets fordiagnosing active protein activity, disease, or predisposition todisease, in a patient having a variant peptide, particularly activitiesand conditions that are known for other members of the family ofproteins to which the present one belongs. Thus, the peptide can beisolated from a biological sample and assayed for the presence of agenetic mutation that results in aberrant peptide. This includes aminoacid substitution, deletion, insertion, rearrangement, (as the result ofaberrant splicing events), and inappropriate post-translationalmodification. Analytic methods include altered electrophoretic mobility,altered tryptic peptide digest, altered protease activity in cell-basedor cell-free assay, alteration in substrate or antibody-binding pattern,altered isoelectric point, direct amino acid sequencing, and any otherof the known assay techniques useful for detecting mutations in aprotein. Such an assay can be provided in a single detection format or amulti-detection format such as an antibody chip array.

In vitro techniques for detection of peptide include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitations andimmunofluorescence using a detection reagent, such as an antibody orprotein binding agent. Alternatively, the peptide can be detected invivo in a subject by introducing into the subject a labeled anti-peptideantibody or other types of detection agent. For example, the antibodycan be labeled with a radioactive marker whose presence and location ina subject can be detected by standard imaging techniques. Particularlyuseful are methods that detect the allelic variant of a peptideexpressed in a subject and methods which detect fragments of a peptidein a sample.

The peptides are also useful in pharmacogenomic analysis.Pharmacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected subjects. See, e.g., Eichelbaum, M. (Clin. Exp.Pharmacol. Physiol. 23(10–11):983–985 (1996)), and Linder, M. W. (Clin.Chem. 43(2):254–266 (1997)). The clinical outcomes of these variationsresult in severe toxicity of therapeutic drugs in certain individuals ortherapeutic failure of drugs in certain individuals as a result ofindividual variation in metabolism. Thus, the genotype of the individualcan determine the way a therapeutic compound acts on the body or the waythe body metabolizes the compound. Further, the activity of drugmetabolizing enzymes affects both the intensity and duration of drugaction. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype. The discovery of genetic polymorphisms in some drugmetabolizing enzymes has explained why some patients do not obtain theexpected drug effects, show an exaggerated drug effect, or experienceserious toxicity from standard drug dosages. Polymorphisms can beexpressed in the phenotype of the extensive metabolizer and thephenotype of the poor metabolizer. Accordingly, genetic polymorphism maylead to allelic protein variants of the protease protein in which one ormore of the protease functions in one population is different from thosein another population. The peptides thus allow a target to ascertain agenetic predisposition that can affect treatment modality. Thus, in aligand-based treatment, polymorphism may give rise to amino terminalextracellular domains and/or other substrate-binding regions that aremore or less active in substrate binding, and protease activation.Accordingly, substrate dosage would necessarily be modified to maximizethe therapeutic effect within a given population containing apolymorphism. As an alternative to genotyping, specific polymorphicpeptides could be identified.

The peptides are also useful for treating a disorder characterized by anabsence of, inappropriate, or unwanted expression of the protein.Accordingly, methods for treatment can include the use of the proteaseprotein or fragments.

Antibodies

The invention also provides antibodies that selectively bind to one ofthe peptides of the present invention, a protein comprising such apeptide, as well as variants and fragments thereof. As used herein, anantibody selectively binds a target peptide when it binds the targetpeptide and does not significantly bind to unrelated proteins. Anantibody is still considered to selectively bind a peptide even if italso binds to other proteins that are not substantially homologous withthe target peptide so long as such proteins share homology with afragment or domain of the peptide target of the antibody. In this case,it would be understood that antibody binding to the peptide is stillselective despite some degree of cross-reactivity.

As used herein, an antibody is defined in terms consistent with thatrecognized within the art: they are multi-subunit proteins produced by amammalian organism in response to an antigen challenge. The antibodiesof the present invention include polyclonal antibodies and monoclonalantibodies, as well as fragments of such antibodies, including, but notlimited to, Fab or F(ab′)₂, and Fv fragments.

Many methods are known for generating and/or identifying antibodies to agiven target peptide. Several such methods are described by Harlow,Antibodies, Cold Spring Harbor Press, (1989).

In general, to generate antibodies, an isolated peptide is used as animmunogen and is administered to a mammalian organism, such as a mouse,rat, rabbit, or monkey. The full-length protein, an antigenic peptidefragment or a fusion protein can be used. Particularly importantfragments are those covering functional domains, and domains of sequencehomology or divergence amongst the family, such as those that canreadily be identified using protein alignment methods and as presentedin FIG. 2.

Antibodies are preferably prepared from regions or discrete fragments ofthe protease proteins. Antibodies can be prepared from any region of thepeptide as described herein. However, preferred regions will includethose involved in function/activity and/or protease/binding partnerinteraction. Sequence alignment, such as that provided in FIG. 2, can beused to identify conserved and unique sequence fragments, and otherparticularly important protein regions.

An antigenic fragment will typically comprise at least 8 contiguousamino acid residues. The antigenic peptide can comprise, however, atleast 10, 12, 14, 16 or more amino acid residues. Such fragments can beselected on a physical property, such as fragments correspond to regionsthat are located on the surface of the protein, e.g., hydrophilicregions or can be selected based on sequence uniqueness.

Detection on an antibody of the present invention can be facilitated bycoupling (i.e., physically linking) the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

Antibody Uses

The antibodies can be used to isolate one of the proteins of the presentinvention by standard techniques, such as affinity chromatography orimmunoprecipitation. The antibodies can facilitate the purification ofthe natural protein from cells and recombinantly produced proteinexpressed in host cells. In addition, such antibodies are useful todetect the presence of one of the proteins of the present invention incells or tissues to determine the pattern of expression of the proteinamong various tissues in an organism and over the course of normaldevelopment. Further, such antibodies can be used to detect protein insitu, in vitro, or in a cell lysate or supernatant in order to evaluatethe abundance and pattern of expression. Also, such antibodies can beused to assess abnormal tissue distribution or abnormal expressionduring development or progression of a biological condition. Antibodydetection of circulating fragments of the full-length protein can beused to identify turnover.

Further, the antibodies can be used to assess expression in diseasestates such as in active stages of the disease or in an individual witha predisposition toward disease related to the protein's function. Whena disorder is caused by an inappropriate tissue distribution,developmental expression, level of expression of the protein, orexpressed/processed form, the antibody can be prepared against thenormal protein. If a disorder is characterized by a specific mutation inthe protein, antibodies specific for this mutant protein can be used toassay for the presence of the specific mutant protein.

The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.The diagnostic uses can be applied, not only in genetic testing, butalso in monitoring a treatment modality. Accordingly, where treatment isultimately aimed at correcting expression level or the presence ofaberrant sequence and aberrant tissue distribution or developmentalexpression, antibodies directed against the protein or relevantfragments can be used to monitor therapeutic efficacy.

Additionally, antibodies are useful in pharmacogenomic analysis. Thus,antibodies prepared against polymorphic proteins can be used to identifyindividuals that require modified treatment modalities. The antibodiesare also useful as diagnostic tools as an immunological marker foraberrant protein analyzed by electrophoretic mobility, isoelectricpoint, tryptic peptide digest, and other physical assays known to thosein the art.

The antibodies are also useful for tissue typing. Thus, where a specificprotein has been correlated with expression in a specific tissue,antibodies that are specific for this protein can be used to identify atissue type.

The antibodies are also useful for inhibiting protein function, forexample, blocking the binding of the protease peptide to a bindingpartner such as a substrate. These uses can also be applied in atherapeutic context in which treatment involves inhibiting the protein'sfunction. An antibody can be used, for example, to block binding, thusmodulating (agonizing or antagonizing) the peptides activity. Antibodiescan be prepared against specific fragments containing sites required forfunction or against intact protein that is associated with a cell orcell membrane.

The invention also encompasses kits for using antibodies to detect thepresence of a protein in a biological sample. The kit can compriseantibodies such as a labeled or labelable antibody and a compound oragent for detecting protein in a biological sample; means fordetermining the amount of protein in the sample; means for comparing theamount of protein in the sample with a standard; and instructions foruse. Such a kit can be supplied to detect a single protein or epitope orcan be configured to detect one of a multitude of epitopes, such as inan antibody detection array. Arrays are described in detail below fornucleic acid arrays and similar methods have been developed for antibodyarrays.

Nucleic Acid Molecules

The present invention further provides isolated nucleic acid moleculesthat encode a protease peptide or protein of the present invention(cDNA, transcript and genomic sequence). Such nucleic acid moleculeswill consist of, consist essentially of, or comprise a nucleotidesequence that encodes one of the protease peptides of the presentinvention, an allelic variant thereof, or an ortholog or paralogthereof.

As used herein, an “isolated” nucleic acid molecule is one that isseparated from other nucleic acid present in the natural source of thenucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. However, there canbe some flanking nucleotide sequences, for example up to about 5 KB, 4KB, 3 KB, 2 KB, or 1 KB or less, particularly contiguous peptideencoding sequences and peptide encoding sequences within the same genebut separated by introns in the genomic sequence. The important point isthat the nucleic acid is isolated from remote and unimportant flankingsequences such that it can be subjected to the specific manipulationsdescribed herein such as recombinant expression, preparation of probesand primers, and other uses specific to the nucleic acid sequences.

Moreover, an “isolated” nucleic acid molecule, such as a transcript/cDNAmolecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or chemicalprecursors or other chemicals when chemically synthesized. However, thenucleic acid molecule can be fused to other coding or regulatorysequences and still be considered isolated.

For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe isolated DNA molecules of the present invention. Isolated nucleicacid molecules according to the present invention further include suchmolecules produced synthetically.

Accordingly, the present invention provides nucleic acid molecules thatconsist of the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1), or anynucleic acid molecule that encodes the protein provided in FIG. 1 (SEQID NO:2). A nucleic acid molecule consists of a nucleotide sequence whenthe nucleotide sequence is the complete nucleotide sequence of thenucleic acid molecule.

The present invention further provides nucleic acid molecules thatconsist essentially of the nucleotide sequence shown in FIG. 1 (SEQ IDNO:1), or any nucleic acid molecule that encodes the protein provided inFIG. 1 (SEQ ID NO:2). A nucleic acid molecule consists essentially of anucleotide sequence when such a nucleotide sequence is present with onlya few additional nucleic acid residues in the final nucleic acidmolecule.

The present invention further provides nucleic acid molecules thatcomprise the nucleotide sequences shown in FIG. 1 (SEQ ID NO:1), or anynucleic acid molecule that encodes the protein provided in FIG. 1 (SEQID NO:2). A nucleic acid molecule comprises a nucleotide sequence whenthe nucleotide sequence is at least part of the final nucleotidesequence of the nucleic acid molecule. In such a fashion, the nucleicacid molecule can be only the nucleotide sequence or have additionalnucleic acid residues, such as nucleic acid residues that are naturallyassociated with it or heterologous nucleotide sequences. Such a nucleicacid molecule can have a few additional nucleotides or can comprisesseveral hundred or more additional nucleotides. A brief description ofhow various types of these nucleic acid molecules can be readilymade/isolated is provided below.

Both coding and non-coding sequences may be encompassed by the presentinvention. For example, the nucleic acid molecules of the presentinvention may contain genomic intronic sequences, 5′ and 3′ non-codingsequences, gene regulatory regions and non-coding intergenic sequences.In general such sequence features can readily be identified usingcomputational tools known in the art. As discussed below, some of thenon-coding regions, particularly gene regulatory elements such aspromoters, are useful for a variety of purposes, e.g., control ofheterologous gene expression, target for identifying gene activitymodulating compounds, and are particularly claimed as fragments of thegenomic sequence provided herein.

The isolated nucleic acid molecules can encode the mature protein plusadditional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature peptide (when the mature form has more than onepeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

As mentioned above, the isolated nucleic acid molecules include, but arenot limited to, the sequence encoding the protease peptide alone, thesequence encoding the mature peptide and additional coding sequences,such as a leader or secretory sequence (e.g., a pre-pro or pro-proteinsequence), the sequence encoding the mature peptide, with or without theadditional coding sequences, plus additional non-coding sequences, forexample introns and non-coding 5′ and 3′ sequences such as transcribedbut non-translated sequences that play a role in transcription, mRNAprocessing (including splicing and polyadenylation signals), ribosomebinding and stability of mRNA. In addition, the nucleic acid moleculemay be fused to a marker sequence encoding, for example, a peptide thatfacilitates purification.

Isolated nucleic acid molecules can be in the form of RNA, such as mRNA,or in the form DNA, including cDNA and genomic DNA obtained by cloningor produced by chemical synthetic techniques or by a combinationthereof. The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

The invention further provides nucleic acid molecules that encodefragments of the peptides of the present invention as well as nucleicacid molecules that encode obvious variants of the protease proteins ofthe present invention that are described above. Such nucleic acidmolecules may be naturally occurring, such as allelic variants (samelocus), paralogs (different locus), and orthologs (different organism),or may be constructed by recombinant DNA methods or by chemicalsynthesis. Such non-naturally occurring variants may be made bymutagenesis techniques, including those applied to nucleic acidmolecules, cells, or organisms. Accordingly, as discussed above, thevariants can contain nucleotide substitutions, deletions, inversions andinsertions. Variation can occur in either or both the coding andnon-coding regions. The variations can produce both conservative andnon-conservative amino acid substitutions.

The present invention further provides non-coding fragments of thenucleic acid molecules provided in the Figures. Preferred non-codingfragments include, but are not limited to, promoter sequences, enhancersequences, gene modulating sequences and gene termination sequences.Such fragments are useful in controlling heterologous gene expressionand in developing screens to identify gene-modulating agents. A promotercan readily be identified as being 5′ to the ATG start site in a genomicsequence.

A fragment comprises a contiguous nucleotide sequence greater than 12 ormore nucleotides. Further, a fragment could at least 30, 40, 50, 100,250 or 500 nucleotides in length. The length of the fragment will bebased on its intended use. For example, the fragment can encodeepitope-bearing regions of the peptide, or can be useful as DNA probesand primers. Such fragments can be isolated using the known nucleotidesequence to synthesize an oligonucleotide probe. A labeled probe canthen be used to screen a cDNA library, genomic DNA library, or mRNA toisolate nucleic acid corresponding to the coding region. Further,primers can be used in PCR reactions to clone specific regions of gene.

A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide pair. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, 20, 25, 40, 50 or moreconsecutive nucleotides.

Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. As described in the Peptide Section,these variants comprise a nucleotide sequence encoding a peptide that istypically 60–70%, 70–80%, 80–90%, and more typically at least about90–95, 96, 97, 98, or 99% homologous to the nucleotide sequence (SEQ IDNO:1) shown in FIG. 1 or a fragment of this sequence. Such nucleic acidmolecules can readily be identified as being able to hybridize undermoderate to stringent conditions, to the nucleotide sequence shown inFIG. 1 or a fragment of the sequence. Allelic variants can readily bedetermined by genetic locus of the encoding gene.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing-underwhich nucleotide sequences encoding a peptide at least 60–70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1–6.3.6. One example ofstringent hybridization conditions are hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45C, followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50–65C. Examples of moderate to lowstringency hybridization conditions are well known in the art.

Nucleic Acid Molecule Uses

The nucleic acid molecules of the present invention are useful forprobes, primers, chemical intermediates, and in biological assays. Thenucleic acid molecules are useful as a hybridization probe for messengerRNA, transcript/cDNA and genomic DNA to isolate full-length cDNA andgenomic clones encoding the peptide shown in FIG. 1 (SEQ ID NO:2) and toisolate cDNA and genomic clones that correspond to variants (alleles,orthologs, etc.) producing the same or related peptides shown in FIG. 1.

The probe can correspond to any sequence along the entire length of thenucleic acid molecules shown in FIG. 1. Accordingly, it could be derivedfrom 5′ noncoding regions, the coding region, and 3′ noncoding regions.However, as discussed, fragments are not to be construed as encompassingfragments disclosed prior to the present invention.

The nucleic acid molecules are also useful as primers for PCR to amplifyany given region of a nucleic acid molecule and are useful to synthesizeantisense molecules of desired length and sequence.

The nucleic acid molecules are also useful for constructing recombinantvectors. Such vectors include expression vectors that express a portionof, or all of, the peptide sequences. Vectors also include insertionvectors, used to integrate into another nucleic acid molecule sequence,such as into the cellular genome, to alter in situ expression of a geneand/or gene product. For example, an endogenous coding sequence can bereplaced via homologous recombination with all or part of the codingregion containing one or more specifically introduced mutations.

The nucleic acid molecules are also useful for expressing antigenicportions of the proteins.

The nucleic acid molecules are also useful as probes for determining thechromosomal positions of the nucleic acid molecules by means of in situhybridization methods.

The nucleic acid molecules are also useful in making vectors containingthe gene regulatory regions of the nucleic acid molecules of the presentinvention.

The nucleic acid molecules are also useful for designing ribozymescorresponding to all, or a part, of the mRNA produced from the nucleicacid molecules described herein.

The nucleic acid molecules are also useful for making vectors thatexpress part, or all, of the peptides.

The nucleic acid molecules are also useful for constructing host cellsexpressing a part, or all, of the nucleic acid molecules and peptides.

The nucleic acid molecules are also useful for constructing transgenicmonkeys or other non-human animals expressing all, or a part, of thenucleic acid molecules and peptides.

The nucleic acid molecules are also useful as hybridization probes fordetermining the presence, level, form and distribution of nucleic acidexpression. Accordingly, the probes can be used to detect the presenceof, or to determine levels of, a specific nucleic acid molecule incells, tissues, and in organisms. The nucleic acid whose level isdetermined can be DNA or RNA. Accordingly, probes corresponding to thepeptides described herein can be used to assess expression and/or genecopy number in a given cell, tissue, or organism. These uses arerelevant for diagnosis of disorders involving an increase or decrease inprotease protein expression relative to normal results.

In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA includes Southern hybridizations and in situhybridization.

Probes can be used as a part of a diagnostic test kit for identifyingcells or tissues that express a protease protein, such as by measuring alevel of a protease-encoding nucleic acid in a sample of cells from asubject e.g., mRNA or genomic DNA, or determining if a protease gene hasbeen mutated.

Nucleic acid expression assays are useful for drug screening to identifycompounds that modulate protease nucleic acid expression.

The invention thus provides a method for identifying a compound that canbe used to treat a disorder associated with nucleic acid expression ofthe protease gene, particularly biological and pathological processesthat are mediated by the protease in cells and tissues that express it.The method typically includes assaying the ability of the compound tomodulate the expression of the protease nucleic acid and thusidentifying a compound that can be used to treat a disordercharacterized by undesired protease nucleic acid expression. The assayscan be performed in cell-based and cell-free systems. Cell-based assaysinclude cells naturally expressing the protease nucleic acid orrecombinant cells genetically engineered to express specific nucleicacid sequences.

The assay for protease nucleic acid expression can involve direct assayof nucleic acid levels, such as mRNA levels, or on collateral compoundsinvolved in the signal pathway. Further, the expression of genes thatare up- or down-regulated in response to the protease protein signalpathway can also be assayed. In this embodiment the regulatory regionsof these genes can be operably linked to a reporter gene such asluciferase.

Thus, modulators of protease gene expression can be identified in amethod wherein a cell is contacted with a candidate compound and theexpression of mRNA determined. The level of expression of protease mRNAin the presence of the candidate compound is compared to the level ofexpression of protease mRNA in the absence of the candidate compound.The candidate compound can then be identified as a modulator of nucleicacid expression based on this comparison and be used, for example totreat a disorder characterized by aberrant nucleic acid expression. Whenexpression of mRNA is statistically significantly greater in thepresence of the candidate compound than in its absence, the candidatecompound is identified as a stimulator of nucleic acid expression. Whennucleic acid expression is statistically significantly less in thepresence of the candidate compound than in its absence, the candidatecompound is identified as an inhibitor of nucleic acid expression.

The invention further provides methods of treatment, with the nucleicacid as a target, using a compound identified through drug screening asa gene modulator to modulate protease nucleic acid expression in cellsand tissues that express the protease. Modulation includes bothup-regulation (i.e. activation or agonization) or down-regulation(suppression or antagonization) or nucleic acid expression.

Alternatively, a modulator for protease nucleic acid expression can be asmall molecule or drug identified using the screening assays describedherein as long as the drug or small molecule inhibits the proteasenucleic acid expression in the cells and tissues that express theprotein.

The nucleic acid molecules are also useful for monitoring theeffectiveness of modulating compounds on the expression or activity ofthe protease gene in clinical trials or in a treatment regimen. Thus,the gene expression pattern can serve as a barometer for the continuingeffectiveness of treatment with the compound, particularly withcompounds to which a patient can develop resistance. The gene expressionpattern can also serve as a marker indicative of a physiologicalresponse of the affected cells to the compound. Accordingly, suchmonitoring would allow either increased administration of the compoundor the administration of alternative compounds to which the patient hasnot become resistant. Similarly, if the level of nucleic acid expressionfalls below a desirable level, administration of the compound could becommensurately decreased.

The nucleic acid molecules are also useful in diagnostic assays forqualitative changes in protease nucleic acid expression, andparticularly in qualitative changes that lead to pathology. The nucleicacid molecules can be used to detect mutations in protease genes andgene expression products such as mRNA. The nucleic acid molecules can beused as hybridization probes to detect naturally occurring geneticmutations in the protease gene and thereby to determine whether asubject with the mutation is at risk for a disorder caused by themutation. Mutations include deletion, addition, or substitution of oneor more nucleotides in the gene, chromosomal rearrangement, such asinversion or transposition, modification of genomic DNA, such asaberrant methylation patterns or changes in gene copy number, such asamplification. Detection of a mutated form of the protease geneassociated with a dysfunction provides a diagnostic tool for an activedisease or susceptibility to disease when the disease results fromoverexpression, underexpression, or altered expression of a proteaseprotein.

Individuals carrying mutations in the protease gene can be detected atthe nucleic acid level by a variety of techniques. Genomic DNA can beanalyzed directly or can be amplified by using PCR prior to analysis.RNA or cDNA can be used in the same way. In some uses, detection of themutation involves the use of a probe/primer in a polymerase chainreaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), suchas anchor PCR or RACE PCR, or, alternatively, in a ligation chainreaction (LCR) (see, e.g., Landegran et al., Science 241:1077–1080(1988); and Nakazawa et al., PNAS 91:360–364 (1994)), the latter ofwhich can be particularly useful for detecting point mutations in thegene (see Abravaya et al., Nucleic Acids Res. 23:675–682 (1995)). Thismethod can include the steps of collecting a sample of cells from apatient, isolating nucleic acid (e.g., genomic, mRNA or both) from thecells of the sample, contacting the nucleic acid sample with one or moreprimers which specifically hybridize to a gene under conditions suchthat hybridization and amplification of the gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. Deletions and insertions can be detected by achange in size of the amplified product compared to the normal genotype.Point mutations can be identified by hybridizing amplified DNA to normalRNA or antisense DNA sequences.

Alternatively, mutations in a protease gene can be directly identified,for example, by alterations in restriction enzyme digestion patternsdetermined by gel electrophoresis.

Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can beused to score for the presence of specific mutations by development orloss of a ribozyme cleavage site. Perfectly matched sequences can bedistinguished from mismatched sequences by nuclease cleavage digestionassays or by differences in melting temperature.

Sequence changes at specific locations can also be assessed by nucleaseprotection assays such as RNase and S1 protection or the chemicalcleavage method. Furthermore, sequence differences between a mutantprotease gene and a wild-type gene can be determined by direct DNAsequencing. A variety of automated sequencing procedures can be utilizedwhen performing the diagnostic assays (Naeve, C. W., (1995)Biotechniques 19:448), including sequencing by mass spectrometry (see,e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv.Chromatogr. 36:127–162 (1996); and Griffin et al., Appl. Biochem.Biotechnol. 38:147–159 (1993)).

Other methods for detecting mutations in the gene include methods inwhich protection from cleavage agents is used to detect mismatched basesin RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242 (1985));Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth. Enzymol.217:286–295 (1992)), electrophoretic mobility of mutant and wild typenucleic acid is compared (Orita et al., PNAS 86:2766 (1989); Cotton etal., Mutat. Res. 285:125–144 (1993); and Hayashi et al., Genet. Anal.Tech. Appl. 9:73–79 (1992)), and movement of mutant or wild-typefragments in polyacrylamide gels containing a gradient of denaturant isassayed using denaturing gradient gel electrophoresis (Myers et al.,Nature 313:495 (1985)). Examples of other techniques for detecting pointmutations include selective oligonucleotide hybridization, selectiveamplification, and selective primer extension.

The nucleic acid molecules are also useful for testing an individual fora genotype that while not necessarily causing the disease, neverthelessaffects the treatment modality. Thus, the nucleic acid molecules can beused to study the relationship between an individual's genotype and theindividual's response to a compound used for treatment (pharmacogenomicrelationship). Accordingly, the nucleic acid molecules described hereincan be used to assess the mutation content of the protease gene in anindividual in order to select an appropriate compound or dosage regimenfor treatment.

Thus nucleic acid molecules displaying genetic variations that affecttreatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

The nucleic acid molecules are thus useful as antisense constructs tocontrol protease gene expression in cells, tissues, and organisms. Anantisense nucleic acid molecule is generally designed to becomplementary to a region of mRNA expressed by the gene so that theantisense molecule can hybridize to the mRNA and thereby blocktranslation of mRNA into protein. Alternatively, an antisense nucleicacid molecule can hybridize to a region of the gene involved intranscription in order to block transcription. Antisense technology iswell established in the art and extensively reviewed in Antisense DrugTechnology: Principles, Strategies, and Applications, Crooke (ed.),Marcel Dekker, Inc.: New York (2001).

Thus, a class of antisense molecules can be used to inactivate mRNA inorder to decrease expression of protease-encoding nucleic acid.Accordingly, these molecules can treat a disorder characterized byabnormal or undesired protease nucleic acid expression. This techniqueinvolves cleavage by means of ribozymes containing nucleotide sequencescomplementary to one or more regions in the mRNA that attenuate theability of the mRNA to be translated. Possible regions include codingregions and particularly coding regions corresponding to the catalyticand other functional activities of the protease protein, such assubstrate binding.

The nucleic acid molecules also provide vectors for gene therapy inpatients containing cells that are aberrant in protease gene expression.Thus, recombinant cells, which include the patient's cells that havebeen engineered ex vivo and returned to the patient, are introduced intoan individual where the cells produce the desired protease protein totreat the individual.

The invention also encompasses kits for detecting the presence of aprotease nucleic acid in a biological sample. For example, the kit cancomprise reagents such as a labeled or labelable nucleic acid or agentcapable of detecting protease nucleic acid in a biological sample; meansfor determining the amount of protease nucleic acid in the sample; andmeans for comparing the amount of protease nucleic acid in the samplewith a standard. The compound or agent can be packaged in a suitablecontainer. The kit can further comprise instructions for using the kitto detect protease protein mRNA or DNA.

Nucleic Acid Arrays

The present invention further provides nucleic acid detection kits, suchas arrays or microarrays of nucleic acid molecules that are based on thesequence information provided in FIG. 1 (SEQ ID NO:1).

As used herein “Arrays” or “Microarrays” refers to an array of distinctpolynucleotides or oligonucleotides synthesized on a substrate, such aspaper, nylon or other type of membrane, filter, chip, glass slide, orany other suitable solid support. In one embodiment, the microarray isprepared and used according to the methods described in U.S. Pat. No.5,837,832, Chee et al., PCT application WO95/11995 (Chee et al.),Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675–1680) and Schena,M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614–10619), all of whichare incorporated herein in their entirety by reference. In otherembodiments, such arrays are produced by the methods described by Brownet al., U.S. Pat. No. 5,807,522.

The microarray or detection kit is preferably composed of a large numberof unique, single-stranded nucleic acid sequences, usually eithersynthetic antisense oligonucleotides or fragments of cDNAs, fixed to asolid support. The oligonucleotides are preferably about 6–60nucleotides in length, more preferably 15–30 nucleotides in length, andmost preferably about 20–25 nucleotides in length. For a certain type ofmicroarray or detection kit, it may be preferable to useoligonucleotides that are only 7–20 nucleotides in length. Themicroarray or detection kit may contain oligonucleotides that cover theknown 5′, or 3′, sequence, sequential oligonucleotides which cover thefull length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray or detection kit may be oligonucleotides that arespecific to a gene or genes of interest.

In order to produce oligonucleotides to a known sequence for amicroarray or detection kit, the gene(s) of interest (or an ORFidentified from the contigs of the present invention) is typicallyexamined using a computer algorithm which starts at the 5′ or at the 3′end of the nucleotide sequence. Typical algorithms will then identifyoligomers of defined length that are unique to the gene, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that may interfere with hybridization. In certainsituations it may be appropriate to use pairs of oligonucleotides on amicroarray or detection kit. The “pairs” will be identical, except forone nucleotide that preferably is located in the center of the sequence.The second oligonucleotide in the pair (mismatched by one) serves as acontrol. The number of oligonucleotide pairs may range from two to onemillion. The oligomers are synthesized at designated areas on asubstrate using a light-directed chemical process. The substrate may bepaper, nylon or other type of membrane, filter, chip, glass slide or anyother suitable solid support.

In another aspect, an oligonucleotide may be synthesized on the surfaceof the substrate by using a chemical coupling procedure and an ink jetapplication apparatus, as described in PCT application WO95/251116(Baldeschweiler et al.) which is incorporated herein in its entirety byreference. In another aspect, a “gridded” array analogous to a dot (orslot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

In order to conduct sample analysis using a microarray or detection kit,the RNA or DNA from a biological sample is made into hybridizationprobes. The mRNA is isolated, and cDNA is produced and used as atemplate to make antisense RNA (aRNA). The aRNA is amplified in thepresence of fluorescent nucleotides, and labeled probes are incubatedwith the microarray or detection kit so that the probe sequenceshybridize to complementary oligonucleotides of the microarray ordetection kit. Incubation conditions are adjusted so that hybridizationoccurs with precise complementary matches or with various degrees ofless complementarity. After removal of nonhybridized probes, a scanneris used to determine the levels and patterns of fluorescence. Thescanned images are examined to determine degree of complementarity andthe relative abundance of each oligonucleotide sequence on themicroarray or detection kit. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large-scale correlationstudies on the sequences, expression patterns, mutations, variants, orpolymorphisms among samples.

Using such arrays, the present invention provides methods to identifythe expression of the protease proteins/peptides of the presentinvention. In detail, such methods comprise incubating a test samplewith one or more nucleic acid molecules and assaying for binding of thenucleic acid molecule with components within the test sample. Suchassays will typically involve arrays comprising many genes, at least oneof which is a gene of the present invention and or alleles of theprotease gene of the present invention.

Conditions for incubating a nucleic acid molecule with a test samplevary. Incubation conditions depend on the format employed in the assay,the detection methods employed, and the type and nature of the nucleicacid molecule used in the assay. One skilled in the art will recognizethat any one of the commonly available hybridization, amplification orarray assay formats can readily be adapted to employ the novel fragmentsof the monkey genome disclosed herein. Examples of such assays can befound in Chard, T, An Introduction to Radioimmunoassay and RelatedTechniques, Elsevier Science Publishers, Amsterdam, The Netherlands(1986); Bullock, G. R. et al., Techniques in Immunocytochemistry,Academic Press, Orlando, Fla. Vol. 1 (1 982), Vol. 2 (1983), Vol. 3(1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays:Laboratory Techniques in Biochemistry and Molecular Biology, ElsevierScience Publishers, Amsterdam, The Netherlands (1985).

The test samples of the present invention include cells, protein ormembrane extracts of cells. The test sample used in the above-describedmethod will vary based on the assay format, nature of the detectionmethod and the tissues, cells or extracts used as the sample to beassayed. Methods for preparing nucleic acid extracts or of cells arewell known in the art and can be readily be adapted in order to obtain asample that is compatible with the system utilized.

In another embodiment of the present invention, kits are provided whichcontain the necessary reagents to carry out the assays of the presentinvention.

Specifically, the invention provides a compartmentalized kit to receive,in close confinement, one or more containers which comprises: (a) afirst container comprising one of the nucleic acid molecules that canbind to a fragment of the monkey genome disclosed herein or anorthologous fragment of the human genome; and (b) one or more othercontainers comprising one or more of the following: wash reagents,reagents capable of detecting presence of a bound nucleic acid.

In detail, a compartmentalized kit includes any kit in which reagentsare contained in separate containers. Such containers include smallglass containers, plastic containers, strips of plastic, glass or paper,or arraying material such as silica. Such containers allows one toefficiently transfer reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated, and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the nucleic acid probe, containers whichcontain wash reagents (such as phosphate buffered saline, Tris-buffers,etc.), and containers which contain the reagents used to detect thebound probe. One skilled in the art will readily recognize that thepreviously unidentified protease gene of the present invention can beroutinely identified using the sequence information disclosed herein canbe readily incorporated into one of the established kit formats whichare well known in the art, particularly expression arrays.

Vectors/Host Cells

The invention also provides vectors containing the nucleic acidmolecules described herein. The term “vector” refers to a vehicle,preferably a nucleic acid molecule, which can transport the nucleic acidmolecules. When the vector is a nucleic acid molecule, the nucleic acidmolecules are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector includes a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

A vector can be maintained in the host cell as an extrachromosomalelement where it replicates and produces additional copies of thenucleic acid molecules. Alternatively, the vector may integrate into thehost cell genome and produce additional copies of the nucleic acidmolecules when the host cell replicates.

The invention provides vectors for the maintenance (cloning vectors) orvectors for expression (expression vectors) of the nucleic acidmolecules. The vectors can function in prokaryotic or eukaryotic cellsor in both (shuttle vectors).

Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the nucleic acid molecules such thattranscription of the nucleic acid molecules is allowed in a host cell.The nucleic acid molecules can be introduced into the host cell with aseparate nucleic acid molecule capable of affecting transcription. Thus,the second nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the nucleic acid molecules from the vector.Alternatively, a trans-acting factor may be supplied by the host cell.Finally, a trans-acting factor can be produced from the vector itself.It is understood, however, that in some embodiments, transcriptionand/or translation of the nucleic acid molecules can occur in acell-free system.

The regulatory sequence to which the nucleic acid molecules describedherein can be operably linked include promoters for directing mRNAtranscription. These include, but are not limited to, the left promoterfrom bacteriophage λ, the lac, TRP, and TAC promoters from E. coli, theearly and late promoters from SV40, the CMV immediate early promoter,the adenovirus early and late promoters, and retrovirus long-terminalrepeats.

In addition to control regions that promote transcription, expressionvectors may also include regions that modulate transcription, such asrepressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. One of ordinary skill in the art would be awareof the numerous regulatory sequences that are useful in expressionvectors. Such regulatory sequences are described, for example, inSambrook et al., Molecular Cloning: A Laboratory Manual. 2nd. ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

A variety of expression vectors can be used to express a nucleic acidmolecule. Such vectors include chromosomal, episomal, and virus-derivedvectors, for example vectors derived from bacterial plasmids, frombacteriophage, from yeast episomes, from yeast chromosomal elements,including yeast artificial chromosomes, from viruses such asbaculoviruses, papovaviruses such as SV40, Vaccinia viruses,adenoviruses, poxviruses, pseudorabies viruses, and retroviruses.Vectors may also be derived from combinations of these sources such asthose derived from plasmid and bacteriophage genetic elements, e.g.cosmids and phagemids. Appropriate cloning and expression vectors forprokaryotic and eukaryotic hosts are described in Sambrook et al.,Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., (1989).

The regulatory sequence may provide constitutive expression in one ormore host cells (i.e. tissue specific) or may provide for inducibleexpression in one or more cell types such as by temperature, nutrientadditive, or exogenous factor such as a hormone or other ligand. Avariety of vectors providing for constitutive and inducible expressionin prokaryotic and eukaryotic hosts are well known to those of ordinaryskill in the art.

The nucleic acid molecules can be inserted into the vector nucleic acidby well-known methodology. Generally, the DNA sequence that willultimately be expressed is joined to an expression vector by cleavingthe DNA sequence and the expression vector with one or more restrictionenzymes and then ligating the fragments together. Procedures forrestriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

The vector containing the appropriate nucleic acid molecule can beintroduced into an appropriate host cell for propagation or expressionusing well-known techniques. Bacterial cells include, but are notlimited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

As described herein, it may be desirable to express the peptide as afusion protein. Accordingly, the invention provides fusion vectors thatallow for the production of the peptides. Fusion vectors can increasethe expression of a recombinant protein, increase the solubility of therecombinant protein, and aid in the purification of the protein byacting for example as a ligand for affinity purification., A proteolyticcleavage site may be introduced at the junction of the fusion moiety sothat the desired peptide can ultimately be separated from the fusionmoiety. Proteolytic enzymes include, but are not limited to, factor Xa,thrombin, and enteroprotease. Typical fusion expression vectors includepGEX (Smith et al., Gene 67:3140 (1988)), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein. Examples of suitableinducible non-fusion E. coli expression vectors include pTrc (Amann etal., Gene 69:301–315 (1988)) and pET 11d (Studier et al., GeneExpression Technology: Methods in Enzymology 185:60–89 (1990)).

Recombinant protein expression can be maximized in host bacteria byproviding a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein. (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 119–128). Alternatively, the sequence ofthe nucleic acid molecule of interest can be altered to providepreferential codon usage for a specific host cell, for example E. coli.(Wada et al., Nucleic Acids Res. 20:2111–2118 (1992)).

The nucleic acid molecules can also be expressed by expression vectorsthat are operative in yeast. Examples of vectors for expression in yeaste.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J. 6:229–234(1987)), pMFa (Kuijan et al., Cell 30:933–943(1982)), pJRY88 (Schultz etal., Gene 54:113–123 (1987)), and pYES2 (Invitrogen Corporation, SanDiego, Calif.).

The nucleic acid molecules can also be expressed in insect cells using,for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9cells) include the pAc series (Smith et al., Mol. Cell Biol. 3:2156–2165(1983)) and the pVL series (Lucklow et al., Virology 170:31–39 (1989)).

In certain embodiments of the invention, the nucleic acid moleculesdescribed herein are expressed in mammalian cells using mammalianexpression vectors. Examples of mammalian expression vectors includepCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman et al., EMBOJ. 6:187–195,(1987)).

The expression vectors listed herein are provided by way of example onlyof the well-known vectors available to those of ordinary skill in theart that would be useful to express the nucleic acid molecules. One ofordinary skill in the art would be aware of other vectors suitable formaintenance propagation or expression of the nucleic acid molecules,described herein. These are found for example in Sambrook, J., Fritsh,E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd,ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1989.

The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the nucleic acid molecule sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

The invention also relates to recombinant host cells containing thevectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to one of ordinary skill in the art. These include, but arenot limited to, calcium phosphate transfection, DEAE-dextran-mediatedtransfection, cationic lipid-mediated transfection, electroporation,transduction, infection, lipofection, and other techniques such as thosefound in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd,ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1989).

Host cells can contain more than one vector. Thus, different nucleotidesequences can be introduced on different vectors of the same cell.Similarly, the nucleic acid molecules can be introduced either alone orwith other nucleic acid molecules that are not related to the nucleicacid molecules such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced or joined tothe nucleic acid molecule vector.

In the case of bacteriophage and viral vectors, these can be introducedinto cells as packaged or encapsulated virus by standard procedures forinfection and transduction. Viral vectors can be replication-competentor replication-defective. In the case in which viral replication isdefective, replication will occur in host cells providing functions thatcomplement the defects.

Vectors generally include selectable markers that enable the selectionof the subpopulation of cells that contain the recombinant vectorconstructs. The marker can be contained in the same vector that containsthe nucleic acid molecules described herein or may be on a separatevector. Markers include tetracycline or ampicillin-resistance genes forprokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

While the mature proteins can be produced in bacteria, yeast, mammaliancells, and other cells under the control of the appropriate regulatorysequences, cell-free transcription and translation systems can also beused to produce these proteins using RNA derived from the DNA constructsdescribed herein.

Where secretion of the peptide is desired, which is difficult to achievewith multi-transmembrane domain containing proteins such as proteases,appropriate secretion signals are incorporated into the vector. Thesignal sequence can be endogenous to the peptides or heterologous tothese peptides.

Where the peptide is not secreted into the medium, which is typicallythe case with proteases, the protein can be isolated from the host cellby standard disruption procedures, including freeze thaw, sonication,mechanical disruption, use of lysing agents and the like. The peptidecan then be recovered and purified by well-known purification methodsincluding ammonium sulfate precipitation, acid extraction, anion orcationic exchange chromatography, phosphocellulose chromatography,hydrophobic-interaction chromatography, affinity chromatography,hydroxylapatite chromatography, lectin chromatography, or highperformance liquid chromatography.

It is also understood that depending upon the host cell in recombinantproduction of the peptides described herein, the peptides can havevarious glycosylation patterns, depending upon the cell, or maybenon-glycosylated as when produced in bacteria. In addition, the peptidesmay include an initial modified methionine in some cases as a result ofa host-mediated process.

Uses of Vectors and Host Cells

The recombinant host cells expressing the peptides described herein havea variety of uses. First, the cells are useful for producing a proteaseprotein or peptide that can be further purified to produce desiredamounts of protease protein or fragments. Thus, host cells containingexpression vectors are useful for peptide production.

Host cells are also useful for conducting cell-based assays involvingthe protease protein or protease protein fragments, such as thosedescribed above as well as other formats known in the art. Thus, arecombinant host cell expressing a native protease protein is useful forassaying compounds that stimulate or inhibit protease protein function.

Host cells are also useful for identifying protease protein mutants inwhich these functions are affected. If the mutants naturally occur andgive rise to a pathology, host cells containing the mutations are usefulto assay compounds that have a desired effect on the mutant proteaseprotein (for example, stimulating or inhibiting function) which may notbe indicated by their effect on the native protease protein.

Genetically engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a monkey or a rodent such as a mouse or rat, inwhich one or more of the cells of the animal include a transgene. Atransgene is exogenous DNA that is integrated into the genome of a cellfrom which a transgenic animal develops and which remains in the genomeof the mature animal in one or more cell types or tissues of thetransgenic animal. These animals are useful for studying the function ofa protease protein and identifying and evaluating modulators of proteaseprotein activity. Other examples of transgenic animals include non-humanprimates, sheep, dogs, cows, goats, chickens, and amphibians.

A transgenic animal can be produced by introducing nucleic acid into themale pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the protease proteinnucleotide sequences can be introduced as a transgene into the genome ofa non-human animal, such as a monkey or mouse.

Any of the regulatory or other sequences useful in expression vectorscan form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the protease protein to particularcells.

Methods for generating non-human transgenic animals via embryomanipulation and microinjection have become conventional in the art andare described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009,both by Leder et al., U.S. Pat. No. 4,873,191 by Wagner et al. and inHogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1986). Similar methods are used forproduction of other transgenic animals. A transgenic founder animal canbe identified based upon the presence of the transgene in its genomeand/or expression of transgenic mRNA in tissues or cells of the animals.A transgenic founder animal can then be used to breed additional animalscarrying the transgene. Moreover, transgenic animals carrying atransgene can further be bred to other transgenic animals carrying othertransgenes. A transgenic animal also includes animals in which theentire animal or tissues in the animal have been produced using thehomologously recombinant host cells described herein.

In another embodiment, transgenic non-human animals can be producedwhich contain selected systems that allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. PNAS 89:6232–6236 (1992).Another example of a recombinase system is the FLP recombinase system ofS. cerevisiae (O'Gorman et al. Science 251:1351–1355 (1991). If acre/loxP recombinase system is used to regulate expression of thetransgene, animals containing transgenes encoding both the Crerecombinase and a selected protein is required. Such animals can beprovided through the construction of “double” transgenic animals, e.g.,by mating two transgenic animals, one containing a transgene encoding aselected protein and the other containing a transgene encoding arecombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut, I. et al. Nature385:810–813 (1997) and PCT International Publication Nos. WO 97/07668and WO 97/07669. In brief, a cell, e.g., a somatic cell, from thetransgenic animal can be isolated and induced to exit the growth cycleand enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell, e.g., the somatic cell, isisolated.

Transgenic animals containing recombinant cells that express thepeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could effect substratebinding, protease protein activity/activation, and signal transduction,may not be evident from in vitro cell-free or cell-based assays.Accordingly, it is useful to provide non-human transgenic animals toassay in vivo protease protein function, including substrateinteraction, the effect of specific mutant protease proteins on proteaseprotein function and substrate interaction, and the effect of chimericprotease proteins. It is also possible to assess the effect of nullmutations, that is, mutations that substantially or completely eliminateone or more protease protein functions.

Knockout Animal Models

The monkey Cathepsin S nucleic acid molecules disclosed herein areuseful for generating “knockout” monkeys, as well as other non-humananimals, in which specific genes are altered, such as to becomeinactivated or inhibited in function or expression. This alteration ofspecific genes is useful for, for example, elucidating specificdisease-associated processes that the gene/encoded protein may beinvolved in, thereby revealing or validating potential therapeutictargets, or for defining the activity of a pharmaceutical compound.

Generally, an animal such as a monkey in which one or more genes orfragments thereof is intentionally altered, such as by deletion ordisruption of the entire gene or a fragment thereof, is referred to as a“knockout” animal. Such alteration can be produced by, for example,homologous recombination or retrovirus integration. Generally, knockoutanimals are produced for the purpose of defining the phenotype of ananimal lacking a particular gene segment (or in which the gene segmentis present but disrupted, such as by insertion of an exogenous nucleicacid-fragment such as a retrovirus, transposon, transgene, a nucleicacid construct or cassette such as an insertion construct as describedbelow, or a nucleic acid construct/cassette having a positive and/ornegative selectable marker such as neomycin resistance, neo), in whichthe function and/or expression of one or more genes may be inactivatedor inhibited. Producing alterations in genes to generate knockoutanimals may also be referred to as gene targeting. An animal in whichone or more fragments of exogenous nucleic acid are introduced into theanimal's genome, whether for the purposes of over-expressing one or moregenes (e.g., by microinjection into the pronucleus of a fertilizedmonkey oocyte, as described above) or for the purpose of disrupting agene to produce a knockout animal (e.g., by insertion of a transgeneinto a target gene in order to inactivate the target gene) may beinterchangeably referred to as a “transgenic” animal, which is furtherdescribed above.

Homologous recombination is commonly used to completely inactivate agene (i.e., to produce a “knockout”). Constructs used for homologousrecombination may be, for example, insertion constructs or replacementconstructs. Insertion constructs contain a region of sequence homologyto the target gene sequence and are introduced by homologousrecombination into the homologous site of the target gene, therebyinterrupting the target gene by adding sequences. In contrast,replacement constructs, which are more commonly used, contain tworegions of homology to the target gene located on either side of thetarget gene sequence. Homologous recombination (by double crossover)then replaces the target gene sequences with the replacement-constructsequences (Current Protocols in Molecular Biology, John Wiley & Sons,Inc., “Manipulating the Mouse Genome”, chapter 23.1, supplement 52–51(2000)).

Gene inactivation by homologous recombination can be achieved in variousways. For example, a region of a gene involved in regulating expressionmay be interrupted (e.g., by introducing an insertion constructcontaining a positive selectable marker), thereby inhibiting orcompletely blocking production of normal mRNA and protein from thetarget gene. As an alternative to interrupting a gene by inserting asequence, a gene may also be inactivated by deleting a portion of thegene or the entire gene. By using a construct having two separateregions of homology to the target gene (e.g., separated by up to about15 kb), the sequences intervening the two homologous regions can bedeleted. Alternatively, small mutations, such as a point mutation, canbe introduced into the target gene sequence. Gene knockouts may also becontrolled either spatially (e.g., in cell type- or tissue-specificknockouts) or temporally by modulating the activity or expression ofrecombinase in a CrelloxP recombinase system (Current Protocols inMolecular Biology, John Wiley & Sons, Inc., “Manipulating the MouseGenome”, chapter 23.1, supplement 52–51 (2000)).

Embryonic stem cell lines, from which knockout non-human animals may beproduced, may be either heterozygous (“single knockout”; described inCurrent Protocols in Molecular Biology, John Wiley & Sons, Inc.,“Manipulating the Mouse Genome”, chapter 23.5, supplements 52–53(2000–2001)) or homozygous (“double knockout”; described in CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc., “Manipulatingthe Mouse Genome”, chapter 23.6, supplements 52–53 (2000–2001)).

Methods for generating knockout non-human animals have becomeconventional in the art and are described in many patents and printedpublications. For example, techniques for high-throughput generation ofknockout mice (referred to as “gene trapping” technology), such as byusing retrovirus integration, are described in U.S. Pat. Nos. 6,218,123;6,207,371; 6,139,833; 6,136,566; and 6,080,576. Production of knockoutmice by homologous recombination is described in Current Protocols inMolecular Biology, John Wiley & Sons, Inc., “Manipulating the MouseGenome”, chapter 23; Robertson, E. J. 1991. “Using embryonic stem cellsto introduce mutations into the mouse germ line.” Biol. Reprod.44:238–245; and Zimmer, A. 1992. “Manipulating the genome by homologousrecombination in embryonic stem cells.” Annu. Rev. Neurosci. 15:115–137.Representative publications reviewing the production and use of knockoutmice include, for example, “Knockout mice: a paradigm shift in modernimmunology.” Mak et al., Nature Rev Immunol 2001 October; 1(1): 1 1–9;“Mouse models for multistep tumorigenesis.”, Wu et al., Trends Cell Biol2001 November; 11(11):S2–9; “Mutant and genetically modified mice asmodels for studying the relationship between aging and carcinogenesis.”Anisimov, Mech Ageing Dev 2001 September;122(12):1221–55; “Unravelinghuman cancer in the mouse: recent refinements to modeling andanalysis.”, Resor et al., Hum Mol Genet 2001 April; 10(7):669–75;“Gene-targeting strategies.” Cheah et al., Methods Mol Biol2000;136:455–63; “Mouse genetics/genomics: an effective approach fordrug target discovery and validation.” West et al., Med Res Rev 2000May;20(3):216–30; “Understanding hypertension through geneticmanipulation in mice.”, Cvetkovic et al., Kidney Int 2000Mar;57(3):863–74; and “Transgenic and gene knockout mice in cancerresearch.”, Viney, Cancer Metastasis Rev 1995 June; 14(2):77–90.

EXAMPLES

Cloning of Monkey Cathepsin S cDNA

Poly-A mRNA was prepared from approximate 2×10⁷ Cynomologous monkeyperipheral blood mononuclear cells (PBMC) using oligo (dT)-celluloseaccording to the manufacturer's instructions (Invitrogen, San Diego,Calif.). The poly-A selected mRNA (0.05 μg) was used to synthesize andamplify a double-stranded cDNA pool using a SMART cDNA synthesis kit(Clontech, Palo Alto, Calif.). The Cynomologous cDNA encoding pro-CatSwas PCR amplified from the cDNA pool using a BD Advantage 2 PCR kit(Clontech, Palo Alto, Calif.) using primers designed according to themonkey (Saimiri boliviensis) pro-CatS sequences [5′ primerCCGGAATTCTTGCATAAAGATCCCACCCTGG (SEQ ID NO:8) and 3′ primerATAGTTTAGCGGCCGCCTAGATTTCTGGGTAAGAGGGG (SEQ ID NO:9)]. The 1 Kbamplified DNA fragment was ligated to a pPIC 9 vector between EcoRI andNot I sites and the nucleotide sequence was determined. The 5′- and3′-un-translated regions, including the pre-sequence of CatS and poly-A,were obtained by PCR amplification using a BD SMART RACE cDNAamplification kit using primers provided by the kit and two genespecific primers [GSP1: GGGATAGGAAGCGTCTGAGTCGATGCCG (SEQ ID NO:10) andGSP2: GGGCCCTGGAAGCACAGCTGAAGC (SEQ ID NO:11)]. The DNA sequencesdetermined from PCR products were used to assemble a full-length CatScDNA (FIG. 1).

Expression of Cathepsin S

The pro-CatS sequence amplified from the cDNA pool was ligated to therestriction sites (EcoRI and Not I) in the multiple cloning site of aPichia expression vector PIC9 to make a signal peptide fusion to thepro-CatS sequence. This expression construct forms a signalpeptide-pro-CatS fusion under a methanol-inducible promoter and allowssecretion of the pro-CatS protein to the culture medium upon removal ofthe signal peptide. The Pro-CatS expression plasmid was transformed intoPichia, and His+ transforments were selected. The expression of pro-CatSin methanol-induced culture medium was further confirmed by Western blotusing human anti-CatS antibody.

Purification of Cathepsin S

Pro-cat S was produced by fermentation of yeast. Cell-free supernatantwas filtered successively through 0.45 μm and 0.22 μm membranes.Filtered supernatant was further concentrated by ultrafiltration anddiafiltrated to auto-activation buffer (50 mM NaOAc, 1 mM EDTA, and 1 mMDTT, pH 4.5). After sitting overnight at 4° C., the mature cat S wascaptured by SP fast flow and followed by separation on source 15S withsalt gradient from 0 to 0.5 M. Fractions containing cat S activity werefurther confirmed by SDS-PAGE and Western blot.

Assay for Cathepsin S

An activity assay for cyno-Cat S was carried out using Z-VVR-AMC assubstrate in a buffer containing 50 mM MES pH6.5, 100 mMNaCl, and 2.5 mMEDTA at room temperature. The reaction volume was 100 μl in amicro-titer reader plate and the RFU was collected over 5 minutes usingan excitation filter of 360nm and an emission filter of 460 nm.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the above-described modesfor carrying out the invention which are obvious to those skilled in thefield of molecular biology or related fields are intended to be withinthe scope of the following claims.

1. An isolated polypeptide, wherein the amino acid sequence of thepolypeptide consists of SEQ ID NO:2.
 2. An isolated polypeptide, whereinthe amino acid sequence of the polypeptide comprises SEQ ID NO:2.
 3. Anisolated polypeptide, wherein the amino acid sequence of saidpolypeptide consists of an amino sequence having at least 99% sequenceidentity to SEQ ID NO:2.
 4. An isolated polypeptide, wherein the aminoacid sequence of said polypeptide comprises an amino acid sequencehaving at least 99% sequence identity to SEQ ID NO:2.
 5. A fusionpolypeptide, wherein the fusion polypeptide comprises the polypeptide ofclaim 2 fused to a heterologous amino add sequence.
 6. A fusionpolypeptide, wherein the fusion polypeptide comprises the polypeptide ofclaim 4 fused to a heterologous amino acid sequence.
 7. A compositioncomprising the polypeptide of claim
 1. 8. A composition comprising thepolypeptide of claim
 2. 9. A composition comprising the polypeptide ofclaim
 3. 10. A composition comprising the polypeptide of claim
 4. 11. Acomposition comprising the polypeptide of claim
 5. 12. A compositioncomprising the polypeptide of claim 6.